Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G.
Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.
Baldwin
Amy E
AE
Department of Chemistry, Pace University, New York, NY, 10038, USA.
Khan
Mateen A
MA
Tumer
Nilgun E
NE
Goss
Dixie J
DJ
Friedland
Diana E
DE
D001665:Binding Sites; D007700:Kinetics; D008954:Models, Biological; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D029604:Phytolacca americana / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D009711:Nucleotides; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D054789:Ribosome Inactivating Proteins, Type 1; C024275:pokeweed antiviral protein